A molecular genetic approach will be used to analyze the structure and function of a promoter region of bacteriophage P22 DNA, in order to determine how promoters direct the binding of RNA polymerase and the initiation of transcription. The particular promoter to be studied (PANT) regulates the expression of two genes which code for regulatory proteins, ant (antirepressor) and arc (antirepressor control). A large number of mutations will be selected which decrease or increase the intrinsic activity of the PANT promoter. In order to determine which nucleotide base pairs are important or essential for promoter function, the nucleotide sequence changes in these mutants will be directly determined by DNA sequencing. Furthermore, the effects of particular nucleotide changes on promoter function will be assayed quantitatively, both in vivo and in vitro. By saturating the promoter with mutations and measuring the effect of each sequence change on kinetics of RNA polymerase binding and transcription initiation, the functional roles of particular nucleotides in the promoter sequence will be determined.